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With the US Food and Drug Administration (FDA) approval of four CD19- and one BCMA-targeted chimeric antigen receptor (CAR) therapy for B cell malignancies, CAR T cell therapy has finally reached the status of a medicinal product. The successful manufacturing of autologous CAR T cell products is a key requirement for this promising treatment modality. By analyzing the composition of 214 apheresis products from 210 subjects across eight disease indications, we found that high CD14+ cell content poses a challenge for manufacturing CAR T cells, especially in patients with non-Hodgkin's lymphoma and multiple myeloma caused by the non-specific phagocytosis of the magnetic beads used to activate CD3+ T cells. We demonstrated that monocyte depletion via rapid plastic surface adhesion significantly reduces the CD14+ monocyte content in the apheresis products and simultaneously boosts the CD3+ content. We established a 40% CD14+ threshold for the stratification of apheresis products across nine clinical trials and demonstrated the effectiveness of this procedure by comparing manufacturing runs in two phase 1 clinical trials. Our study suggests that CD14+ content should be monitored in apheresis products, and that the manufacturing of CAR T cells should incorporate a step that lessens the CD14+ cell content in apheresis products containing more than 40% to maximize the production success.

Chimeric antigen receptor (CAR) T-cell therapy has shown remarkable responses in B-cell malignancies. However, many patients suffer from limited response and tumor relapse due to lack of persisting CAR T cells and immune escape. These clinical challenges have compromised the long-term efficacy of CAR T-cell therapy and call for the development of novel CAR designs. We demonstrated that CAR T cells secreting a cytokine interleukin-36γ (IL-36γ) showed significantly improved CAR T-cell expansion and persistence, and resulted in superior tumor eradication compared with conventional CAR T cells. The enhanced cellular function by IL-36γ was mediated through an autocrine manner. In addition, activation of endogenous antigen-presenting cells (APCs) and T cells by IL-36γ aided the formation of a secondary antitumor response, which delayed the progression of antigen-negative tumor challenge. Together, our data provide preclinical evidence to support the translation of this design for an improved CAR T-cell-mediated antitumor response.

Chimeric Antigen Receptor (CAR)-T cells represent a major breakthrough in cancer therapy, wherein a patient’s own T cells are engineered to recognize a tumor antigen, resulting in activation of a local cytotoxic immune response. However, CAR-T cell therapies are currently limited to the treatment of B-cell cancers and their effectiveness is hindered by resistance from antigen-negative tumor cells, immunosuppression in the tumor microenvironment, eventual exhaustion of T-cell immunologic functions, and frequent severe toxicities. To overcome these problems, we have developed a novel class of CAR-T cells engineered to express an enzyme that activates a systemically-administered small-molecule prodrug in situ at a tumor site. We show that these Synthetic Enzyme-Armed KillER (SEAKER) cells exhibit enhanced anticancer activity with small-molecule prodrugs, both in vitro and in vivo in mouse tumor models. This modular platform enables combined targeting of cellular and small-molecule therapies to treat cancers and potentially a variety of other diseases.

Immunosuppressive tumor microenvironment (TME) and ascites-derived spheroids in ovarian cancer (OC) facilitate tumor growth and progression, and also pose major obstacles for cancer therapy. The molecular pathways involved in the OC-TME interactions, how the crosstalk impinges on OC aggression and chemoresistance are not well-characterized. Here, we demonstrate that tumor-derived UBR5, an E3 ligase overexpressed in human OC associated with poor prognosis, is essential for OC progression principally by promoting tumor-associated macrophage recruitment and activation via key chemokines and cytokines. UBR5 is also required to sustain cell-intrinsic β-catenin-mediated signaling to promote cellular adhesion/colonization and organoid formation by controlling the p53 protein level. OC-specific targeting of UBR5 strongly augments the survival benefit of conventional chemotherapy and immunotherapies. This work provides mechanistic insights into the novel oncogene-like functions of UBR5 in regulating the OC-TME crosstalk and suggests that UBR5 is a potential therapeutic target in OC treatment for modulating the TME and cancer stemness., Ovarian cancer cells often metastasize to the peritoneal cavity, forming spheroid-like structures and promoting a highly immunosuppressive tumor microenvironment. Here, the authors show that the ubiquitin ligase UBR5 is required for ovarian cancer growth and metastasis, sustaining spheroid formation and the infiltration of immunosuppressive tumor associated macrophages.

While effective in specific settings, adoptive chimeric antigen receptor (CAR) T cell therapy for cancer requires further improvement and optimization. Our previous results show that CD40L-overexpressing CAR T cells mobilize endogenous immune effectors, resulting in improved antitumor immunity. However, the cell populations required for this protective effect remain to be identified. Here we show, by analyzing Batf3−/− mice lacking the CD103+ conventional dendritic cell type 1 (cDC1) subpopulation important for antigen cross-presentation, that CD40L-overexpressing CAR T cells elicit an impaired antitumor response in the absence of cDC1s. We further find that CD40L-overexpressing CAR T cells stimulate tumor-resident CD11b−CD103− double-negative (DN) cDCs to proliferate and differentiate into cDC1s in wild-type mice. Finally, re-challenge experiments show that endogenous CD8+ T cells are required for protective antitumor memory in this setting. Our findings thus demonstrate the stimulatory effect of CD40L-overexpressing CAR T cells on innate and adaptive immune cells, and provide a rationale for using CD40L-overexpressing CAR T cells to improve immunotherapy responses., CD40L-expressing chimeric antigen receptor (CAR) T cells show enhanced anti-tumor immunity, but the cellular mechanisms are still unclear. Here we show, by analyzing mice deficient of conventional dendritic cell type 1 (cDC1) that cDC1s are induced by CD40L+ CAR T cells to prime endogenous CD8 T cells for a stronger anti-tumor immune response.

Factors contributing to hematopoietic recovery following chimeric antigen receptor (CAR) T-cell therapy have not been well studied. In an analysis of 83 patients with hematologic malignancies treated with CAR T-cell therapy, we describe patterns of hematopoietic recovery and evaluate potentially associated factors. We included patients who received axicabtagene ciloleucel (n = 30) or tisagenlecleucel (n = 10) for B-cell lymphoma, CD19-28z CAR T therapy for B-cell acute lymphoblastic leukemia (NCT01044069; n = 37), or B-cell maturation antigen targeting CAR T cells for multiple myeloma (NCT03070327; n = 6). Patients treated with CAR T cells who had not progressed, died, or received additional chemotherapy had “recovered” (per definition in Materials and methods section) hemoglobin, platelet, neutrophil, and white blood cell counts at rates of 61%, 51%, 33%, and 28% at month 1 postinfusion and 93%, 90%, 80%, and 59% at month 3 postinfusion, respectively. Univariate analysis showed that increasing grade of immune effector cell–associated neurological syndrome (ICANS), baseline cytopenias, CAR construct, and higher peak C-reactive protein or ferritin levels were statistically significantly associated with a lower likelihood of complete count recovery at 1 month; a similar trend was seen for cytokine release syndrome (CRS). After adjustment for baseline cytopenia and CAR construct, grade ≥3 CRS or ICANS remained significantly associated with the absence of complete count recovery at 1 month. Higher levels of vascular endothelial growth factor and macrophage-derived chemokines, although not statistically significant, were seen patients without complete count recovery at 1 month. This remains to be studied further in larger prospective studies.

Replication-competent retrovirus (RCR) is a safety concern for individuals treated with retroviral gene therapy. RCR detection assays are used to detect RCR in manufactured vector, transduced cell products infused into research subjects, and in the research subjects after treatment. In this study, we reviewed 286 control (n = 4) and transduced cell products (n = 282) screened for RCR in the National Gene Vector Biorepository. The transduced cell samples were submitted from 14 clinical trials. All vector products were previously shown to be negative for RCR prior to use in cell transduction. After transduction, all 282 transduced cell products were negative for RCR. In addition, 241 of the clinical trial participants were also screened for RCR by analyzing peripheral blood at least 1 month after infusion, all of which were also negative for evidence of RCR infection. The majority of vector products used in the clinical trials were generated in the PG13 packaging cell line. The findings suggest that screening of the retroviral vector product generated in PG13 cell line may be sufficient and that further screening of transduced cells does not provide added value. Keywords: retrovirus, safety testing, replicating virus, lentivirus

T-cell receptor (TCR)–modified T-cell gene therapy can target a variety of extracellular and intracellular tumor-associated antigens, yet has had little clinical success. A potential explanation for limited antitumor efficacy is a lack of T-cell activation in vivo. We postulated that expression of proinflammatory cytokines in TCR-modified T cells would activate T cells and enhance antitumor efficacy. We demonstrate that expression of interleukin 18 (IL18) in tumor-directed TCR-modified T cells provides a superior proinflammatory signal than expression of interleukin 12 (IL12). Tumor-targeted T cells secreting IL18 promote persistent and functional effector T cells and a proinflammatory tumor microenvironment. Together, these effects augmented overall survival of mice in the pmel-1 syngeneic tumor model. When combined with sublethal irradiation, IL18-secreting pmel-1 T cells were able to eradicate tumors, whereas IL12-secreting pmel-1 T cells caused toxicity in mice through excessive cytokine secretion. In another xenograft tumor model, IL18 secretion enhanced the persistence and antitumor efficacy of NY-ESO-1–reactive TCR-modified human T cells as well as overall survival of tumor-bearing mice. These results demonstrate a rationale for optimizing the efficacy of TCR-modified T-cell cancer therapy through expression of IL18. See related commentary by Wijewarnasuriya et al., p. 732

Multiple myeloma (MM) remains generally incurable, calling for the development of novel treatment strategies such as chimeric antigen receptor (CAR) T cell therapy. Most clinically tested CAR T cell therapies for MM target B cell maturation antigen (BCMA), but despite high response rates, many patients relapse (Raje N. NEJM 2019). BCMA negative-low MM cells are implicated as a reservoir preceding relapse (Brudno J. JCO 2018; Cohen A. JCI 2019). Our aims are to (1) evaluate whether upfront simultaneous targeting of an additional antigen such as G protein-coupled receptor class C group 5 member D (GPRC5D; Smith EL. Sci Trans Med 2019) can mitigate BCMA escape-mediated relapse in MM, and (2) compare dual targeting strategies to identify an optimal approach. Dual targeting for CD19/CD22 malignancies has been investigated, and multiple approaches are feasible; however, approaches have yet to be comprehensively compared head to head. Here, we compare 2 parallel production and 3 single-vector dual targeting strategies (Fig. 1A). To enhance clinical translatability, all strategies are built on the BCMA(125)/4-1BBζ CAR (BCMA scFv 125; Smith EL. Mol Ther 2018), which is currently under multi-center clinical investigation (NCT03430011; Mailankody S. ASH 2018). We confirmed that all dual targeted approaches lyse, proliferate, and secrete polyfunctional cytokines specifically in response to BCMA and GPRC5D mono- and dual-positive cell lines and/or primary patient MM aspirate samples. Activity in vivo was confirmed using the bone marrow-tropic OPM2 MM model (endogenously BCMA+GPRC5D+). In all experiments MM cells (2 x 106) were injected IV into NSG mice and engrafted/expanded for 14 days before treatment. A high dose of all dual targeted CAR T cell approaches (3 x 106 CAR+) induced long-term disease control (median overall survival (mOS) BCMA(125) non-signaling del control 32d vs other groups mOS not reached; p < 0.05). Prevention of latent BCMA escape-mediated relapse was evaluated by re-challenge of previously treated long-surviving mice with 2 x 106 OPM2 BCMA CRISPR KO (OPM2BCMA KO) cells at day 100 without re-treatment. While mice previously treated with BCMA(125)/41BBζ CAR T cells succumbed to OPM2BCMA KO disease, dual targeted approaches prevented OPM2BCMA KO growth (mOS BCMA mono-targeted arm 37d post re-challenge vs other groups mOS not reached; p < 0.05). To better recapitulate human MM and distinguish among dual targeting approaches, we modeled established BCMA heterogeneous disease by spiking 5-10% OPM2BCMA KO into bulk OPM2WT cells for injection. Each OPM2 population was modified to express distinct luciferases for simultaneous in vivo monitoring by bioluminescent imaging (BLI). Treatment with a moderate (5 x 105) dose of CAR T cells eradicated OPM2WT cells in all groups, but anti-GPRC5D CARs with CD28 co-stimulation, whether included within a mixed T cell population or in a bicistronic construct (Fig. 1A ii, iv), failed to control OPM2BCMA KO cells (Fig. 1B). Correspondingly, 4-1BB-only containing CAR T cells had increased in vivo expansion (2.1-4.1-fold increase CAR T cell BLI at day 7 over CD28 containing groups; p < 0.05). As this result is likely from greater activation-induced cell death in the CD28-containing approaches that was not rescued by 4-1BB, we later compared 4-1BB-only containing approaches (Fig. 1A i, iii, v). These 3 dual targeting approaches effectively controlled OPM2WT disease at moderate (1 x 106 CAR+) and low (2.5 x 105 CAR+) doses. However, when using a sub-therapeutic dose (2.5 x 105 CAR+) in the OPM2BCMA KO-spiked model, the tandem scFv-single stalk design was least effective in controlling OPM2BCMA KO disease (Fig 1C). At a dose that is sub-therapeutic to control OPM2WT disease (1 x 105 CAR+), the bicistronic dual 4-1BB design (Fig. 1A iii) was more effective in eradicating tumor compared with the parallel production approach (6-fold difference tumor BLI at day 28; p < 0.05). These results indicate that upfront dual targeting of BCMA/GPRC5D with CAR T cells can mitigate BCMA escape-mediated relapse in a model of MM. While parallel infusion of separate BCMA- and GPRC5D-targeted CAR T cells is effective, a single bicistronic vector encoding two 4-1BB-containing CARs avoids the practical challenges of parallel manufacturing, and uniquely may provide superior anti-MM efficacy. Figure Disclosures Fernandez de Larrea: Takeda: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding. Brentjens:JUNO Therapeutics: Consultancy, Patents & Royalties, Research Funding; Celgene: Consultancy. Smith:Celgene: Consultancy, Patents & Royalties, Research Funding; Fate Therapeutics and Precision Biosciences: Consultancy.

High-dose chemotherapy followed by autologous stem cell transplantation (HDT-ASCT) is the standard of care for relapsed or chemorefractory diffuse large B-cell lymphoma (rel/ref DLBCL). Only 50% of patients are cured with this approach. We investigated whether CD19-specific chimeric antigen receptor (CAR) T cells administered following HDT-ASCT may enhance progression-free survival (PFS). Methods: Eligibility for this study includes poor-risk rel/ref aggressive B-NHL chemosensitive to salvage therapy with: 1) FDG-PET (+) or 2) bone marrow involvement. Patients underwent BEAM conditioned HDT-ASCT and followed by 19-28z CAR-T cells on days +2 and +3. Results: Of 15 subjects treated on study, dose-limiting toxicity was observed at both dose levels (5 x106 and 1 x107 19-28z CAR-T/kg). Ten of 15 subjects experienced CAR T cell-induced neurotoxicity and/or cytokine-release syndrome (CRS), which were associated with greater CAR T cell persistence (p=0.05) but not peak CAR T cell expansion. Serum IFN-g elevation (pl0.001) and possibly IL-10 (p=0.07) were associated with toxicity. The 2-year PFS is 30% (95% CI: 20-70%). Two subjects with progression of disease (POD) were CD19 (-) on re-biopsy. Subjects given decreased naive-like (CD45RA+CCR7+) CD4+ and CD8+ CAR T cells experienced superior PFS (p=0.02 and 0.04, respectively). There was no association between CAR T cell peak expansion, persistence or cytokine changes and PFS. Conclusions: 19-28z CAR T cells following HDT-ASCT was associated with a high-incidence of reversible neurotoxicity and CRS. Following HDT-ASCT, effector CD4+ and CD8+ immunophenotypes may improve disease control. Phenotype selection and/or multiple infusions may be the focus of the next clinical trial. This study is registered at www.clinicaltrials.gov as #NCT01840566.

We present a case of a patient with multiply relapsed, refractory myeloma whose clinical course showed evidence of a synergistic abscopal-like response to chimeric antigen receptor (CAR) T-cell therapy and localized radiotherapy (XRT). Shortly after receiving B-cell maturation antigen (BCMA)–targeted CAR T-cell therapy, the patient required urgent high-dose steroids and XRT for spinal cord compression. Despite the steroids, the patient had a durable systemic response that could not be attributed to XRT alone. Post-XRT findings included a second wave of fever and increased CRP and IL6, beginning 21 days after CAR T cells, which is late for cytokine-release syndrome from CAR T-cell therapy alone on this trial. Given this response, which resembled cytokine-release syndrome, immediately following XRT, we investigated changes in the patient's T-cell receptor (TCR) repertoire over 10 serial time points. Comparing T-cell diversity via Morisita's overlap indices (CD), we discovered that, although the diversity was initially stable after CAR T-cell therapy compared with baseline (CD = 0.89–0.97, baseline vs. 4 time points after CAR T cells), T-cell diversity changed after the conclusion of XRT, with >30% newly expanded TCRs (CD = 0.56–0.69, baseline vs. 4 time points after XRT). These findings suggest potential synergy between radiation and CAR T-cell therapies resulting in an abscopal-like response.

CD19-directed chimeric antigen receptor (CAR) T cells are clinically effective in a limited set of leukemia patients. However, CAR T-cell therapy thus far has been largely restricted to targeting extracellular tumor-associated antigens (TAA). Herein, we report a T-cell receptor-mimic (TCRm) CAR, termed WT1-28z, that is reactive to a peptide portion of the intracellular onco-protein Wilms Tumor 1(WT1), as it is expressed on the surface of the tumor cell in the context of HLA-A*02:01. T cells modified to express WT1-28z specifically targeted and lysed HLA-A*02:01+ WT1+ tumors and enhanced survival of mice engrafted with HLA-A*02:01+, WT1+ leukemia or ovarian tumors. This in vivo functional validation of TCRm CAR T cells provides the proof-of-concept necessary to expand the range of TAA that can be effectively targeted for immunotherapy to include attractive intracellular targets, and may hold great potential to expand on the success of CAR T-cell therapy.

Summary Chimeric antigen receptor (CAR) T cells provide great efficacy in B cell malignancies. However, improved CAR T cell therapies are still needed. Here, we engineered tumor-targeted CAR T cells to constitutively express the immune-stimulatory molecule CD40 ligand (CD40L) and explored efficacy in different mouse leukemia/lymphoma models. We observed that CD40L+ CAR T cells circumvent tumor immune escape via antigen loss through CD40/CD40L-mediated cytotoxicity and induction of a sustained, endogenous immune response. After adoptive cell transfer, the CD40L+ CAR T cells displayed superior antitumor efficacy, licensed antigen-presenting cells, enhanced recruitment of immune effectors, and mobilized endogenous tumor-recognizing T cells. These effects were absent in Cd40−/− mice and provide a rationale for the use of CD40L+ CAR T cells in cancer treatment.

Patients with residual chronic lymphocytic leukemia (CLL) following initial purine analog-based chemoimmunotherapy exhibit a shorter duration of response and may benefit from novel therapeutic strategies. We and others have previously described the safety and efficacy of autologous T cells modified to express anti-CD19 chimeric antigen receptors (CARs) in patients with relapsed or refractory B cell acute lymphoblastic leukemia and CLL. Here we report the use of CD19-targeted CAR T cells incorporating the intracellular signaling domain of CD28 (19-28z) as a consolidative therapy in 8 patients with residual CLL following first-line chemoimmunotherapy with pentostatin, cyclophosphamide, and rituximab. Outpatients received low-dose conditioning therapy with cyclophosphamide (600 mg/m(2)), followed by escalating doses of 3 × 10(6), 1 × 10(7), or 3 × 10(7) 19-28z CAR T cells/kg. An objective response was observed in 3 of 8 patients (38%), with a clinically complete response lasting more than 28 months observed in two patients. Self-limited fevers were observed post-CAR T cell infusion in 4 patients, contemporaneous with elevations in interleukin-6 (IL-6), IL-10, IL-2, and TGF-α. None developed severe cytokine release syndrome or neurotoxicity. CAR T cells were detectable post-infusion in 4 patients, with a longest observed persistence of 48 days by qPCR. Further strategies to enhance CAR T cell efficacy in CLL are under investigation.

B cell maturation antigen (BCMA) has recently been identified as an important multiple myeloma (MM)-specific target for chimeric antigen receptor (CAR) T cell therapy. In CAR T cell therapy targeting CD19 for lymphoma, host immune anti-murine CAR responses limited the efficacy of repeat dosing and possibly long-term persistence. This clinically relevant concern can be addressed by generating a CAR incorporating a human single-chain variable fragment (scFv). We screened a human B cell-derived scFv phage display library and identified a panel of BCMA-specific clones from which human CARs were engineered. Despite a narrow range of affinity for BCMA, dramatic differences in CAR T cell expansion were observed between unique scFvs in a repeat antigen stimulation assay. These results were confirmed by screening in a MM xenograft model, where only the top preforming CARs from the repeat antigen stimulation assay eradicated disease and prolonged survival. The results of this screening identified a highly effective CAR T cell therapy with properties, including rapid in vivo expansion (>10,000-fold, day 6), eradication of large tumor burden, and durable protection to tumor re-challenge. We generated a bicistronic construct including a second-generation CAR and a truncated-epithelial growth factor receptor marker. CAR T cell vectors stemming from this work are under clinical investigation.

Chimeric antigen receptors (CARs) are recombinant receptors composed of an antibody-derived extracellular single-chain variable fragment linked to a costimulatory signaling domain combined with the...

Adoptive cellular therapy (ACT) is now considered a bona fide treatment modality within the evolving field of anticancer immunotherapy. Great advances have enabled the adoptive transfer of tumour-selective lymphocytes for the treatment of a variety of malignancies. Unfortunately, this selectivity has led to the emergence of antigen-loss variants. New strategies need to be employed to minimize the incidence of this phenomenon, enabling the full potential of ACT to be realized.

International audience; The HVEM (TNFRSF14) receptor gene is among the most frequently mutated genes in germinal center lymphomas. We report that loss of HVEM leads to cell-autonomous activation of B cell proliferation and drives the development of GC lymphomas in vivo. HVEM-deficient lymphoma B cells also induce a tumor-supportive microenvironment marked by exacerbated lymphoid stroma activation and increased recruitment of T follicular helper (T-FH) cells. These changes result from the disruption of inhibitory cell-cell interactions between the HVEM and BTLA (B and T lymphocyte attenuator) receptors. Accordingly, administration of the HVEM ectodomain protein (solHVEM ((P37-V202))) binds BTLA and restores tumor suppression. To deliver solHVEM to lymphomas in vivo, we engineered CD19-targeted chimeric antigen receptor (CAR) T cells that produce solHVEM locally and continuously. These modified CAR-T cells show enhanced therapeutic activity against xenografted lymphomas. Hence, the HVEM-BTLA axis opposes lymphoma development, and our study illustrates the use of CAR-T cells as "micro-pharmacies'' able to deliver an anti-cancer protein.

The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor-armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms.CART-19 yield 70% response rates in patients with B-ALL, but also produce escape variants. We discovered that the underlying mechanism is the selection for preexisting alternatively spliced CD19 isoforms with the compromised CART-19 epitope. This mechanism suggests a possibility of targeting alternative CD19 ectodomains, which could improve survival of patients with B-cell neoplasms.

Disease relapse or progression is a major cause of death following umbilical cord blood (UCB) transplantation (UCBT) in patients with high-risk, relapsed or refractory acute lymphoblastic leukemia (ALL). Adoptive transfer of donor-derived T cells modified to express a tumor-targeted chimeric antigen receptor (CAR) may eradicate persistent disease after transplantation. Such therapy has not been available to UCBT recipients, however, due to the low numbers of available UCB T cells and the limited capacity for ex vivo expansion of cytolytic cells. We have developed a novel strategy to expand UCB T cells to clinically relevant numbers in the context of exogenous cytokines. UCB-derived T cells cultured with interleukin (IL)-12 and IL-15 generated >150-fold expansion with a unique central memory/effector phenotype. Moreover, UCB T cells were modified to both express the CD19-specific CAR, 1928z, and secrete IL-12. 1928z/IL-12 UCB T cells retained a central memory-effector phenotype and had increased antitumor efficacy in vitro. Furthermore, adoptive transfer of 1928z/IL-12 UCB T cells resulted in significantly enhanced survival of CD19(+) tumor-bearing SCID-Beige mice. Clinical translation of CAR-modified UCB T cells could augment the graft-versus-leukemia effect after UCBT and thus further improve disease-free survival of transplant patients with B-cell ALL.

CD19-targeted chimeric antigen receptor (CAR) T cells are currently being tested in the clinic with very promising outcomes. However, limitations to CAR T cell therapy exist. These include lack of efficacy against some tumors, specific targeting of tumor cells without affecting normal tissue and retaining activity within the suppressive tumor microenvironment. Whilst promising clinical trials are in progress, preclinical development is focused on optimizing CAR design, to generate “armored CAR T cells” which are protected from the inhibitory tumor microenvironment. Studies investigating the expression of cytokine transgenes, combination therapy with small molecule inhibitors or monoclonal antibodies are aimed at improving the anti-tumor efficacy of CAR T cell therapy. Other strategies aimed at improving CAR T cell therapy include utilizing dual CARs and chemokine receptors to more specifically target tumor cells. This review will describe the current clinical data and some novel “armored CAR T cell” approaches for improving anti-tumor efficacy therapy.

Adults with relapsed B cell acute lymphoblastic leukemia (B-ALL) have a dismal prognosis. Only those patients able to achieve a second remission with no minimal residual disease (MRD) have a hope for long-term survival in the context of a subsequent allogeneic hematopoietic stem cell transplantation (allo-HSCT). We have treated five relapsed B-ALL subjects with autologous T cells expressing a CD19-specific CD28/CD3ζ second-generation dual-signaling chimeric antigen receptor (CAR) termed 19-28z. All patients with persistent morphological disease or MRD(+) disease upon T cell infusion demonstrated rapid tumor eradication and achieved MRD(-) complete remissions as assessed by deep sequencing polymerase chain reaction. Therapy was well tolerated, although significant cytokine elevations, specifically observed in those patients with morphologic evidence of disease at the time of treatment, required lymphotoxic steroid therapy to ameliorate cytokine-mediated toxicities. Indeed, cytokine elevations directly correlated to tumor burden at the time of CAR-modified T cell infusions. Tumor cells from one patient with relapsed disease after CAR-modified T cell therapy, who was ineligible for additional allo-HSCT or T cell therapy, exhibited persistent expression of CD19 and sensitivity to autologous 19-28z T cell-mediated cytotoxicity, which suggests potential clinical benefit of additional CAR-modified T cell infusions. These results demonstrate the marked antitumor efficacy of 19-28z CAR-modified T cells in patients with relapsed/refractory B-ALL and the reliability of this therapy to induce profound molecular remissions, forming a highly effective bridge to potentially curative therapy with subsequent allo-HSCT.

T cells can be genetically modified to target tumors through the expression of a chimeric antigen receptor (CAR). Most notably, CAR T cells have demonstrated clinical efficacy in hematologic malignancies with more modest responses when targeting solid tumors. However, CAR T cells also have the capacity to elicit expected and unexpected toxicities including: cytokine release syndrome, neurologic toxicity, “on target/off tumor” recognition, and anaphylaxis. Theoretical toxicities including clonal expansion secondary to insertional oncogenesis, graft versus host disease, and off-target antigen recognition have not been clinically evident. Abrogating toxicity has become a critical step in the successful application of this emerging technology. To this end, we review the reported and theoretical toxicities of CAR T cells and their management.

The ability of immune-competent donor T cells to mediate a beneficial graft-versus-leukemia (GVL) effect was first identified in the setting of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for hematologic malignancies. Unfortunately, with the exception of chronic myelogenous leukemia and EBV-induced lymphoproliferative disease, allo-HSCT GVL lacks the potency to significantly affect disease progression or recurrence in most other hematologic malignancies. The inadequacy of a GVL effect using past approaches is particularly evident in patients with lymphoid malignancies. However, with the advent of improved gene transfer technology, genetically modified tumor-specific immune effectors have extended cellular immunotherapy to lymphoid malignancies. One promising strategy entails the introduction of genes encoding artificial receptors called chimeric antigen receptors (CARs), which redirect the specificity and function of immune effectors. CAR-modified T cells targeted to the B cell–specific CD19 antigen have demonstrated promising results in multiple early clinical trials, supporting further investigation in patients with B-cell cancers. However, disparities in clinical trial design and CAR structure have complicated the discovery of the optimal application of this technology. Recent preclinical studies support additional genetic modifications of CAR-modified T cells to achieve optimal clinical efficacy using this novel adoptive cellular therapy.

Background: Xenograft mouse models represent helpful tools for preclinical studies on human tumors. For modeling the complexity of the human disease, primary tumor cells are by far superior to established cell lines. As qualified exemplary model, patients’ acute lymphoblastic leukemia cells reliably engraft in mice inducing orthotopic disseminated leukemia closely resembling the disease in men. Unfortunately, disease monitoring of acute lymphoblastic leukemia in mice is hampered by lack of a suitable readout parameter. Design and Methods: Patients’ acute lymphoblastic leukemia cells were lentivirally transduced to express the membranebound form of Gaussia luciferase. In vivo imaging was established in individual patients’ leukemias and extensively validated. Results: Bioluminescence in vivo imaging enabled reliable and continuous follow-up of individual mice. Light emission strictly correlated to post mortem quantification of leukemic burden and revealed a logarithmic, time and cell number dependent growth pattern. Imaging conveniently quantified frequencies of leukemia initiating cells in limiting dilution transplantation assays. Upon detecting a single leukemia cell within more than 10,000 bone marrow cells, imaging enabled monitoring minimal residual disease, time to tumor re-growth and relapse. Imaging quantified therapy effects precisely and with low variances, discriminating treatment failure from partial and complete responses. Conclusions: For the first time, we characterized in detail how in vivo imaging reforms preclinical studies on patient-derived tumors upon increasing monitoring resolution. In the future, in vivo imaging will enable performing precise preclinical studies on a broad range of highly demanding clinical challenges, such as treatment failure, resistance in leukemia initiating cells, minimal residual disease and relapse.

Purpose: Most patients diagnosed with ovarian cancer will ultimately die from their disease. For this reason, novel approaches to the treatment of this malignancy are needed. Adoptive transfer of a patient's own T cells, genetically modified ex vivo through the introduction of a gene encoding a chimeric antigen receptor (CAR) targeted to a tumor-associated antigen, is a novel approach to the treatment of ovarian cancer. Experimental Design: We have generated several CARs targeted to the retained extracellular domain of MUC16, termed MUC-CD, an antigen expressed on most ovarian carcinomas. We investigate the in vitro biology of human T cells retrovirally transduced to express these CARs by coculture assays on artificial antigen-presenting cells as well as by cytotoxicity and cytokine release assays using the human MUC-CD+ ovarian tumor cell lines and primary patient tumor cells. Further, we assess the in vivo antitumor efficacy of MUC-CD–targeted T cells in SCID-Beige mice bearing peritoneal human MUC-CD+ tumor cell lines. Results: CAR-modified, MUC-CD–targeted T cells exhibited efficient MUC-CD–specific cytolytic activity against both human ovarian cell and primary ovarian carcinoma cells in vitro. Furthermore, expanded MUC-CD–targeted T cells infused through either i.p. injection or i.v. infusion into SCID-Beige mice bearing orthotopic human MUC-CD+ ovarian carcinoma tumors either delayed progression or fully eradicated disease. Conclusion: These promising preclinical studies justify further investigation of MUC-CD–targeted T cells as a potential therapeutic approach for patients with high-risk MUC16+ ovarian carcinomas. Clin Cancer Res; 16(14); 3594–606. ©2010 AACR.